Crystal structure of glutaminyl cyclase

ABSTRACT

Novel crystal structures of human and murine glutaminyl cyclase (QC, EC 2.3.2.5), methods of preparing the crystals, as well as the use of said crystal structures for identifying inhibitors of human and murine glutaminyl cyclase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Division of U.S. application Ser. No. 13/213,507, filed Aug. 19, 2011. This application claims priority to U.S. Provisional Application Ser. No. 61/375,103, filed on Aug. 19, 2010. U.S. application Ser. No. 13/213,507 and 61/375,103 are incorporated herein by reference in their entireties to the extent permitted by law.

MATERIAL INCORPORATED-BY-REFERENCE

The Sequence Listing, which is a part of the present disclosure, includes a computer readable form comprising nucleotide and/or amino acid sequences of the present disclosure. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to the novel crystal structure of glutaminyl cyclase (QC, EC 2.3.2.5). QC catalyzes the intramolecular cyclization of N-terminal glutamine residues into pyroglutamic acid (5-oxo-prolyl, pGlu*) under liberation of ammonia and the intramolecular cyclization of N-terminal glutamate residues into pyroglutamic acid under liberation of water.

BACKGROUND OF THE INVENTION

Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the intramolecular cyclization of N-terminal glutamine residues into pyroglutamic acid (pGlu*) liberating ammonia. A QC was first isolated by Messer from the latex of the tropical plant Carica papaya in 1963 (Messer, M. 1963 Nature 4874, 1299). 24 years later, a corresponding enzymatic activity was discovered in animal pituitary (Busby, W. H. J. et al. 1987 J Biol Chem 262, 8532-8536; Fischer, W. H. and Spiess, J. 1987 Proc Natl Acad Sci USA 84, 3628-3632). For the mammalian QC, the conversion of Gln into pGlu by QC could be shown for the precursors of TRH and GnRH (Busby, W. H. J. et al. 1987 J Biol Chem 262, 8532-8536; Fischer, W. H. and Spiess, J. 1987 Proc Natl Acad Sci USA 84, 3628-3632). In addition, initial localization experiments of QC revealed a co-localization with its putative products of catalysis in bovine pituitary, further improving the suggested function in peptide hormone synthesis (Bockers, T. M. et al. 1995 J Neuroendocrinol 7, 445-453). In contrast, the physiological function of the plant QC is less clear. In the case of the enzyme from C. papaya, a role in the plant defense against pathogenic microorganisms was suggested (El Moussaoui, A. et al. 2001 Cell Mol Life Sci 58, 556-570). Putative QCs from other plants were identified by sequence comparisons recently (Dahl, S. W. et al. 2000 Protein Expr Purif 20, 27-36). The physiological function of these enzymes, however, is still ambiguous.

The QCs known from plants and animals show a strict specificity for L-Glutamine in the N-terminal position of the substrates and their kinetic behavior was found to obey the Michaelis-Menten equation (Pohl, T. et al. 1991 Proc Natl Acad Sci USA 88, 10059-10063; Consalvo, A. P. et al. 1988 Anal Biochem 175, 131-138; Gololobov, M. Y. et al. 1996 Biol Chem Hoppe Seyler 377, 395-398). A comparison of the primary structures of the QCs from C. papaya and that of the highly conserved QC from mammals, however, did not reveal any sequence homology (Dahl, S. W. et al. 2000 Protein Expr Purif 20, 27-36). Whereas the plant QCs appear to belong to a new enzyme family (Dahl, S. W. et al. 2000 Protein Expr Purif 20, 27-36), the mammalian QCs were found to have a pronounced sequence homology to bacterial aminopeptidases (Bateman, R. C. et al. 2001 Biochemistry 40, 11246-11250), leading to the conclusion that the QCs from plants and animals have different evolutionary origins.

Recently, it was shown that recombinant human QC as well as QC-activity from brain extracts catalyze both, the N-terminal glutaminyl as well as glutamate cyclization. Most striking is the finding, that cyclase-catalyzed Glu₁-conversion is favored around pH 6.0 while Gln₁-conversion to pGlu-derivatives occurs with a pH-optimum of around 8.0. Since the formation of pGlu-Aβ-related peptides can be suppressed by inhibition of recombinant human QC and QC-activity from pig pituitary extracts, the enzyme QC is a target in drug development for treatment of Alzheimer's disease.

U.S. Pat. No. 7,572,614 (Wang et al) and Huang et al (2005) PNAS 102(37), 13117-13122 both describe one example of the crystal structure of soluble glutaminyl cyclase. The crystal structure disclosed in Wang et al and Huang et al was generated using a protein expressed in E. coli, which results in a lack of glycosylation. It is well known that all mammalian QC contain at least one glycosylation site (Pohl, T. et al. (1991) Proc Natl Acad Sci USA 88, 10059-10063; Song, I. et al. (1994) J Mol Endocrinol 13, 77-86), which is glycosylated in the QC crystallized according to the present disclosure by virtue of being expressed in eukaryotic hosts, which can be observed in the crystal structures presented herein. In addition, all mammalian QCs contain two conserved cysteine residues close to the active site, which form a disulfide bond. In the crystal structure of Wang et al and Huang et al, the disulfide bond is lacking. The expression of mammalian secretory proteins in bacteria frequently results in the absence of disulfide formation (Hannig, G. and Makrides, S. C. (1998) Trends Biotechnol 16, 54-60). The disulfide bond is clearly present in both the human and murine QC crystal structures presented herein. Notably, the mutational analyses provided in the examples described herein suggest an important stabilizing function of the disulfide bond upon the overall structure. Furthermore, in the structure of Wang et al and Huang et al, a segment of residues (L205-H206-W207) close to the active site appears in two different conformations. Due to the orientations, the binding mode of substrates is affected and reliable mechanistic conclusions could not be drawn (Huang et al., 2005). The residue W207 is conserved in mammalian QCs (W208 in murine QC). In the human and murine QC crystal structures presented herein, the orientation is identical, although the adjacent residues L205 and H206 are not conserved. Therefore, the structural orientation of residues appears non-native in the crystal structure of Wang et al and Huang et al.

The expression of the murine and human QC in an eukaryotic host, as described in the present disclosure, allows the crystallization and structural refinement of a native mammalian QC and, importantly, unambiguous determination of the binding modes of inhibitors, as exemplified by the structural resolution of murine QC with three different inhibitory compounds (listed in Table 1 as Inhibitor A, Inhibitor B and Inhibitor C), which have similar inhibitory potency between human and murine QC.

In contrast to the structures described in previous publications by Wang et al and Huang et al., the present disclosure shows that the post-translational modifications disulfide formation and glycosylation lead to a single structural arrangement of the residues L205-H206-W207 in human and murine QC. The residues have a direct effect on the binding mode of active-site directed compounds. The multiple orientations of W207 in previous studies, led to variances in the binding modes of active-site-directed compounds.

TABLE 1 K_(i)-values [μM] of selected inhibitors for murine (mQC) and human QC (hQC) Inhibitor mQC hQC Inhibitor A 0.173  0.0542 Inhibitor B n.d. 0.0613 Inhibitor C 0.0513 0.106

In contrast, unambigous binding modes of compounds and orientations of W207 were observed with three different inhibitors and even in a structure without inhibitor bound. Thus, the methods as described in the present disclosures provide substantial advances for structure-driven drug design of QC inhibitors.

The conclusion is particularly strengthened by the structural assessment of QCs from two different mammals.

SUMMARY OF THE INVENTION

According to a first embodiment there is provided a crystal comprising human glutaminyl cyclase having a characterised space group of C121 and unit cell dimensions of +/−5% of a=82.6 Å, b=63.9 Å, c=77.5 Å, α=90.0°, β=105.7° and γ=90.0°.

According to a second embodiment there is provided a crystal comprising murine glutaminyl cyclase having a characterised space group of P212121 and having unit cell dimensions of +/−5% of a=42.7 Å, b=84.0 Å and c=96.5 Å.

According to a third embodiment there is provided a method of preparing the crystal of human glutaminyl cyclase as described herein, which comprises the steps of:

-   -   (a) providing a solution of human glutaminyl cyclase, optionally         in the presence of a known glutaminyl cyclase inhibitor, in a         suitable buffer such as 25 mM Bis-Tris pH6.8/100 mM NaCl buffer;     -   (b) mixing the solution with a crystallization solution         comprising 30% (v/v) 2-methyl 2,4-pentanediol and 11% (w/v)         4000PEG; and     -   (c) incubating the mixture under conditions to promote hanging         drop vapor diffusion for a time sufficient to produce the         crystal of human glutaminyl cyclase.

According to a further embodiment there is provided a crystal comprising human glutaminyl cyclase obtainable by the crystallisation method as defined herein.

According to a fourth embodiment there is provided a method of preparing the crystal of murine glutaminyl cyclase as described herein, which comprises the steps of:

-   -   (a) providing a solution of murine glutaminyl cyclase,         optionally in the presence of a known glutaminyl cyclase         inhibitor, in a suitable buffer such as 15 mM Bis-Tris pH6.8/100         mM NaCl buffer;     -   (b) mixing the solution with a crystallization solution         comprising 0.1M sodium acetate pH 5.3, 0.2M ammonium sulphate         and 12% (w/v) 2000 MME-PEG; and     -   (c) incubating the mixture under conditions to promote hanging         drop vapor diffusion for a time sufficient to produce the         crystal of murine glutaminyl cyclase.

According to a further embodiment there is provided a crystal comprising murine glutaminyl cyclase obtainable by the crystallisation method as defined herein.

According to a fifth embodiment there is provided a method of identifying an inhibitor of human glutaminyl cyclase which comprises the following steps:

-   -   (a) generating a 3-dimensional model of human glutaminyl cyclase         using the structural coordinates described in FIG. 1;     -   (b) analysing the binding pocket provided by residues E202, D159         and H330 of SEQ ID NO: 1 according to the coordinates of FIG. 1;     -   (c) performing computer modeling analysis to identify an         inhibitor compound which may associate with the binding pocket         of human glutaminyl cyclase.

According to a sixth embodiment there is provided a method of identifying an inhibitor of murine glutaminyl cyclase which comprises the following steps:

-   -   (a) generating a 3-dimensional model of murine glutaminyl         cyclase using the structural coordinates described in any of         FIGS. 2 to 4;     -   (b) analysing the binding pocket provided by residues D160, E203         and H331 of SEQ ID NO: 13 according to the coordinates of FIGS.         2 to 4;     -   (c) performing computer modeling analysis to identify an         inhibitor compound which may associate with the binding pocket         of murine glutaminyl cyclase.

Other objects and features will be in part apparent and in part pointed out hereinafter.

DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIGS. 1-1 to 1-41 describes the X-ray coordinates of human QC crystallized with 0.1M imidazol pH 8, wherein “RES” and “#” correspond to the amino acid residue and number of the amino acid in SEQ ID NO: 1.

FIGS. 2-1 to 2-45 describes the X-ray coordinates of murine QC crystallized with QC inhibitor Inhibitor A, wherein “RES” and “#” correspond to the amino acid residue and number of the amino acid in SEQ ID NO: 13.

FIGS. 3-1 to 3-46 describes the X-ray coordinates of murine QC crystallized with QC inhibitor Inhibitor B, wherein “RES” and “#” correspond to the amino acid residue and number of the amino acid in SEQ ID NO: 13.

FIGS. 4-1 to 4-45 describes the X-ray coordinates of murine QC crystallized with QC inhibitor Inhibitor C, wherein “RES” and “#” correspond to the amino acid residue and number of the amino acid in SEQ ID NO: 13.

FIG. 5A describes the results of SDS-PAGE analysis of hQC WT.

FIG. 5B describes the results of SDS-PAGE analysis of the C139A/C164A mutant.

FIG. 6 describes the CD spectra of the far UV region of hQC WT and the C139A/C164A mutant at 20° C.

FIG. 7 describes temperature dependence of the mean residue ellipticity (θ_(MRW)) of hQC WT and the C139A/C164A mutant at a wavelength of 227 nm.

FIG. 8A describes CD spectra of the far UV region of hQC WT at increasing temperature.

FIG. 8B describes CD spectra of the far UV region of the C139A/C164A mutant at increasing temperature.

FIG. 9A describes fluorescence emission spectra in a wavelength range between 320 and 400 nm of hCQ WT at different GdmCl concentrations.

FIG. 9B describes fluorescence emission spectra wavelength range between 320 and 400 nm of C139A/C164A mutant at different GdmCl concentrations.

FIG. 10 describes dependence of the relative fluorescence from the GdmCl concentration of hQC WT and the C139A/C164A mutant at a wavelength of 337 nm.

FIG. 11A-B demonstrates photographic representations of the crystals of human glutaminyl cyclase grown in buffer consisting of 0.1M imidazol pH 8, 30% (v/v) 2-methyl 2,4-pentanediol and 11% (w/v) 4000PEG.

FIG. 12A describes a cartoon representation of the obtained three dimensional structure of the human glutaminyl cyclase described by the X-ray coordinates shown in FIG. 1. They are shown in light gray 3-sheet structures, in dark gray a helices and in green the random coiled loops.

FIG. 12B shows an orthogonal orientation of FIG. 12A.

FIG. 13 demonstrates an overlay of the electron density at 1.5 sigma level with the modeled residues building the active site. With a stick representation are displayed in the lightest gray the active site related residues D¹⁵⁹, E²⁰² and H³³⁰ and in medium gray an imidazole molecule (see ring structure middle left of figure) coordinating the catalytic zinc cation which is shown with a ball representation in the center of the figure.

FIG. 14 demonstrates a photographic representation of examples of the crystals of the mouse glutaminyl cyclase grown in buffer consisting of 0.1M sodium acetate pH 5.3 0.2M ammonium sulphate 12% (w/v) 2000 MME-PEG and 1 mM Inhibitor A.

FIG. 15A describes a cartoon representation of the obtained three dimensional structure of the mouse glutaminyl cyclase described by the X-ray coordinates shown in FIGS. 2-4. In dark gray 3-sheet in Light gray α-helices and in gray strands the random coiled structures are shown.

FIG. 15B shows an orthogonal orientation of FIG. 15A.

FIG. 16 demonstrates an overlay of the electron density at 1.5 sigma level with the modeled residues building the active centre. With a stick representation are displayed in medium gray the active site related residues D¹⁶⁰, E²⁰³ and H³³¹ (see left upper, left middle, and left lower structures inside of tips) and in light gray the inhibitor molecule Inhibitor A (see right structure) coordinating the catalytic zinc cation shown with a ball representation in grey (see middle of figure).

FIG. 17 demonstrates an overlay of the electron density at 1.5 sigma level with the modeled residues building the disulfide bridge. With a stick representation are displayed the residues involved in the disulfide bridge, C¹⁴⁰ and C¹⁶⁵, formation and in cartoon representation are displayed the secondary structures surrounding that disulfide bridge.

FIG. 18 demonstrates an overlay of the electron density at 1.5 sigma level with the modeled residues building the active centre. With a stick representation are displayed in medium gray the active site related residues D¹⁶⁰, E²⁰³ and H³³¹ (see left middle, left upper, and left lower structures) and in light gray the inhibitor molecule Inhibitor C (see right middle structure) coordinating the catalytic zinc cation shown with a sphere representation in grey (see just left of center of figure) and in ball representation in dark gray are shown water molecules (see small ball structures upper middle and upper right of figure).

FIG. 19 demonstrates an overlay of the electron density at 1.5 sigma level with the modeled residues building the active centre. With a stick representation are displayed in medium gray the active site related residues D¹⁶⁰, E²⁰³ and H³³¹ (see left upper, left middle, and left lower structures) and in light to medium gray the inhibitor molecule Inhibitor B (see right middle structure) coordinating the catalytic zinc cation shown with a sphere representation in grey (see left of center of figure) and in ball representation in dark gray are shown water molecules (see upper right and middle right of figure).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to crystals of human and murine glutaminyl cyclase, where the crystals are of sufficient quality and size to allow for the determination of the three-dimensional X-ray diffraction structure of glutaminyl cyclase to a resolution of about 1.8 angstrom and to about 2.1 angstroms in C121 and P212121 space groups, respectively. The present disclosure also relates to methods for preparing and crystallizing human and murine glutaminyl cyclase. The crystals of human and murine glutaminyl cyclase, as well as information derived from their crystal structures, can be used to analyze and modify glutaminyl cyclase as well as to identify compounds that interact with glutaminyl cyclase.

Thus, according to a first embodiment, there is provided a crystal comprising human glutaminyl cyclase having a characterised space group of C121 and unit cell dimensions of +/−5% of a=82.6 Å, b=63.9 Å, c=77.5 Å, α=90.0°, β=105.7° and γ=90.0°.

In an embodiment, the crystal can have unit cell dimensions of a=82.6 Å, b=63.9 Å and c=77.5 Å.

In an embodiment, the crystal can have unit cell dimensions of α=90.0°, β=105.7° and γ=90.0°.

In an embodiment, the crystal can diffract x-rays for determination of atomic co-ordinates of the crystal to a resolution of between 2.08 Å and 11.98 Å.

According to a second embodiment there is provided a crystal comprising murine glutaminyl cyclase having a characterised space group of P212121 and having unit cell dimensions of +1-5% of a=42.7 Å, b=84.0 Å, c=96.5 Å, α=90.0°, β=90.0° and γ=90.0°.

In an embodiment, the crystal can have unit cell dimensions of a=42.7 Å, b=83.0 Å, c=95.7 Å, α=90.0°, β=90.0° and γ=90.0°. This embodiment relates to the crystal structure of murine glutaminyl cyclase complexed with a glutaminyl cyclase inhibitor, referred to herein as Inhibitor A, which has the structure as follows:

Inhibitor A can inhibit both human and mouse QC in vitro and moreover, Inhibitor A is soluble in water and therefore useful for crystallization.

In an embodiment, the crystal can have unit cell dimensions of a=42.7 Å, b=84.6 Å, c=96.5 Å, α=90.0°, β=90.0° and γ=90.0°. This embodiment relates to the crystal structure of murine glutaminyl cyclase complexed with a glutaminyl cyclase inhibitor, referred to herein as Inhibitor B, which has the structure as follows:

In an embodiment, the crystal can have unit cell dimensions of a=42.7 Å, b=84.6 Å, c=97.2 Å, α=90.0°, β=90.0° and γ=90.0°. This embodiment relates to the crystal structure of murine glutaminyl cyclase complexed with a glutaminyl cyclase inhibitor, referred to herein as Inhibitor C, which has the structure as follows:

In an embodiment, the crystal can have unit cell dimensions of α=90.0°, β=90.0° and γ=90.0°.

In an embodiment, the crystal can diffract x-rays for determination of atomic co-ordinates of the crystal to a resolution of between 1.9 Å and 32.0 Å.

In an embodiment, the crystal can diffract x-rays for determination of atomic co-ordinates of the crystal to a resolution of between 1.8 Å and 19.81 Å. This embodiment relates to the resolution of the crystal structure of murine glutaminyl cyclase complexed with a glutaminyl cyclase inhibitor, referred to herein as Inhibitor A.

In an embodiment, the crystal can diffract x-rays for determination of atomic co-ordinates of the crystal to a resolution of between 1.98 Å and 32.0 Å. This embodiment relates to the resolution of the crystal structure of murine glutaminyl cyclase complexed with a glutaminyl cyclase inhibitor, referred to herein as Inhibitor B.

In an embodiment, the crystal can diffract x-rays for determination of atomic co-ordinates of the crystal to a resolution of between 1.9 Å and 30.7 Å. This embodiment relates to the resolution of the crystal structure of murine glutaminyl cyclase complexed with a glutaminyl cyclase inhibitor, referred to herein as Inhibitor C.

It will be appreciated that the crystals within the scope of the present disclosure can include both apo crystals and co-crystals. The apo crystals generally comprise substantially pure glutaminyl cyclase. The co-crystals generally comprise substantially pure glutaminyl cyclase with a binding ligand, such as a glutaminyl cyclase inhibitor, bound to glutaminyl cyclase. Thus, in some embodiments, there is provided a co-crystal comprising the crystal as defined herein bound to a binding ligand, such as a glutaminyl cyclase inhibitor.

In an embodiment, the human glutaminyl cyclase can comprise or consist of amino acid residues A35 to L361 of SEQ ID NO:1.

In an embodiment, the murine glutaminyl cyclase can comprise or consist of amino acid residues A36 to L362 of SEQ ID NO:13.

It will be appreciated that the crystals comprising glutaminyl cyclase are not limited to those obtainable from naturally occurring or native glutaminyl cyclase. The crystals can include mutants that have one or more amino acid insertions, deletions, or substitutions in native glutaminyl cyclase. Therefore, mutants of native glutaminyl cyclase can be obtained by replacing at least one (such as up to 10, e.g. up to 25) amino acid residue in a native glutaminyl cyclase with a different amino acid residue, or by adding or deleting amino acid residues within the native protein or at the N- or C-terminus of the native protein, and can have substantially the same three-dimensional structure as native glutaminyl cyclase from which the mutant is derived.

By having substantially the same three-dimensional structure is meant as having a set of atomic structure coordinates from an apo- or co-crystal that have a root mean square deviation of less than or equal to about 2 Å when superimposed with the atomic structure coordinates of native glutaminyl cyclase from which the mutant is derived when at least about 50% to about 100% of the alpha carbon atoms of native glutaminyl cyclase are included in the superposition.

In some instances, it may be particularly advantageous or convenient to substitute, delete, or add amino acid residues to native glutaminyl cyclase in order to provide convenient cloning sites in the cDNA encoding the protein, to aid in protein purification, and the like. Such substitutions, deletions, or additions, which do not substantially alter the three dimensional structure of native glutaminyl cyclase, will be apparent to those skilled in the art.

It should be noted that the mutant polypeptides contemplated herein need not exhibit glutaminyl cyclase activity. Indeed, amino acid substitutions, additions, or deletions that interfere with the activity of glutaminyl cyclase but which do not significantly alter the three-dimensional structure of glutaminyl cyclase can also be included. Such polypeptide crystals, or the atomic structure coordinates obtained therefrom, can be used to identify compounds that bind to native glutaminyl cyclase and which may affect the activity of the native glutaminyl cyclase.

The derivative crystals of the present disclosure generally comprise glutaminyl cyclase crystals in non-covalent/covalent association with one or more metal atoms. The polypeptide may correspond to native or mutated glutaminyl cyclase. One such example of a suitable metal atom is zinc.

The co-crystals of glutaminyl cyclase generally comprise crystals comprising glutaminyl cyclase in association with one or more compounds bound to glutaminyl cyclase. The association may be covalent or non-covalent. In one embodiment, the compounds bound to glutaminyl cyclase comprise glutaminyl cyclase inhibitors. Examples of such glutaminyl cyclase inhibitors include those described in WO 2004/098625, WO 2004/098591, WO 2005/039548, WO 2005/075436, WO 2008/055945, WO 2008/055947, WO 2008/055950 and WO 2008/065141.

The native and mutated glutaminyl cyclase described herein may be isolated from natural sources or produced by methods well known to those skilled in the art of molecular biology. Detailed experimental for the preparation of human and murine glutaminyl cyclase is described in Examples 1 and 2 herein, respectively.

The apo, derivative and co-crystals of glutaminyl cyclase can be obtained by techniques well-known in the art of protein crystallography, including batch, liquid bridge, dialysis, vapor diffusion, such as hanging drop vapor diffusion, and the like (See for example, McPherson, 1982, Preparation and Analysis of Protein Crystals, John Wiley, NY; McPherson, 1990, Eur. J. Biochem. 189:1-23; Webber, 1991, Adv. Protein Chem. 41:1-36; Crystallization of Nucleic Acids and Proteins, Edited by Ducruix and Giege, Oxford University Press; Protein Crystallization Techniques, Strategies, and Tips, Edited by Bergfors, International University Line, 1999).

In an embodiment, glutaminyl cyclase crystals, apo or co-crystals can be grown by vapor diffusion, such as hanging drop vapor diffusion.

In an embodiment, there is provided a method of preparing the crystal of human glutaminyl cyclase as described herein, which comprises the steps of:

-   -   (a) providing a solution of human glutaminyl cyclase, optionally         in the presence of a known glutaminyl cyclase inhibitor, in a         suitable buffer such as 25 mM Bis-Tris pH6.8/100 mM NaCl buffer;     -   (b) mixing the solution with a crystallization solution         comprising 30% (v/v) 2-methyl 2,4-pentanediol and 11% (w/v)         4000PEG; and     -   (c) incubating the mixture under conditions to promote hanging         drop vapor diffusion for a time sufficient to produce the         crystal of human glutaminyl cyclase.

In an embodiment, there is provided a method of preparing the co-crystal of human glutaminyl cyclase bound to a binding ligand, such as a glutaminyl cyclase inhibitor, which comprises the steps of:

-   -   (a) providing a solution of human glutaminyl cyclase in the         presence of a binding ligand, such as a glutaminyl cyclase         inhibitor, in a suitable buffer such as 25 mM Bis-Tris pH6.8/100         mM NaCl buffer;     -   (b) mixing the solution with a crystallization solution         comprising 30% (v/v) 2-methyl 2,4-pentanediol and 11% (w/v)         4000PEG; and     -   (c) incubating the mixture under conditions to promote hanging         drop vapor diffusion for a time sufficient to produce the         co-crystal of human glutaminyl cyclase bound to a binding         ligand, such as a glutaminyl cyclase inhibitor.

In an embodiment, there is provided a crystal or co-crystal comprising human glutaminyl cyclase obtainable by the crystallisation method as defined herein.

Crystallization of human glutaminyl cyclase to produce co-crystals can be carried out as described below and in Example 1. As described, purified human glutaminyl cyclase is concentrated to 10 mg/mL in the presence of a suitable buffer, such as 25 mM Bis-Tris pH6.8/100 mM NaCl buffer and the ligand imidazol is added to a final concentration of 0.1 M. Macroscopic plate crystal forms are grown by hanging drop vapor diffusion at 21° C. by mixing an equal volume of protein solution with a crystallization solution comprising 30% (v/v) 2-methyl 2,4-pentanediol and 11% (w/v) 4000PEG. Crystals typically appeared 10-15 days after the experiment was initiated and examples of such crystals obtained following this protocol are shown in FIG. 11. It will be appreciated that to produce apo-crystals, the ligand is omitted in the above protocol.

The human glutaminyl cyclase crystals may be frozen prior to data collection. The crystals can be cryo-protected with for example, either (a) 20-30% saturated glucose present in the crystallization setup, (b) ethanol added to 15-20%, (c) ethylene glycol added to 10-20% and PEG10,000 brought up to 25%, or (d) glycerol added to 15%. In an embodiment, the crystals are cryo-protected with the addition of glycerol added to 15% (v/v). The crystals can be either briefly immersed in the cryo-protectant or soaked in the cryo-protectant for periods as long as a day. Freezing can be accomplished by immersing the crystal in a bath of liquid nitrogen or by placing the crystal in a stream of nitrogen gas at −180° C.

As described in Example 1, the crystal structure of human glutaminyl cyclase in complex with imidazole was obtained. A summary of the crystal's attributes for human glutaminyl cyclase bound to imidazole are listed in Table 3 and the three dimensional structure coordinates for space groups C121 for human glutaminyl cyclase bound to imidazole are shown in FIG. 1.

References herein to “coordinates” include references to Cartesian coordinates derived from the mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of X-ray by the atoms of a protein or protein complex in crystal form. The diffraction data are used to calculate an electron density map of the repeating units of the crystal, The electron density maps are then used to establish the positions of the individual atoms of the molecule or molecular complex.

Ribbon and overlay diagrams of human glutaminyl cyclase bound to imidazole based upon the coordinates for space groups C121 are shown in FIGS. 12 and 13, respectively. In particular, it was found that the protein comprised a globular α/β hydrolase fold. A central β-sheet was formed with six β-strands all in parallel fashion but not the second. This β-sheet was observed to be surrounded by α-helices in a sandwich manner with two helices in one side and six more α-helices in the opposite face. The protein's structure was completed with a rather large amount of random coiled loops which are believed to build the active site of the enzyme.

References herein to “active site” include references to a specific region (or atom) in a molecular entity that is capable of entering into a stabilising interaction with another molecular entity. In certain embodiments, the term also refers to the reactive parts of a macromolecule that directly participate in its specific combination with another molecule. In an alternative embodiment, a binding site may be comprised or defined by the three dimensional arrangement of one or more amino acid residues within a folded polypeptide. References to “binding pocket” shall be interpreted in an analogous manner to “active site” and it will be appreciated that these terms may be used interchangeably.

This active site of human glutaminyl cyclase was found to accommodate a zinc ion which is coordinated by three protein residues, E²⁰², D¹⁵⁹ and H³³⁰. Thus, in an embodiment, the crystal can comprise a binding pocket provided by residues E202, D159 and H330 of SEQ ID NO: 1 according to the coordinates of FIG. 1.

Moreover, the protein shows the presence of a disulfide bridge between residues C¹³⁹ and C¹⁶⁴. Thus, in an embodiment, the crystal can comprise a disulfide bridge between residues C139 and C164 of SEQ ID NO: 1. As discussed hereinbefore, the presence of such a disulfide bridge has not previously been reported during crystallisation studies with QC (Wang et al, Huang et al).

Additionally four cis-peptide bonds are present between the pair of residues: D¹⁵⁹-S¹⁶⁰, H²²⁸—P²²⁹, G³⁰¹-V³⁰² and S³²³—P³²⁴. Thus, in an embodiment, the crystal can comprise one or more cis-peptide bonds between any of the following pairs of residues: D¹⁵⁹-S¹⁶⁰, H²²⁸—P²²⁹, G³⁰¹-V³⁰² and S³²³—P³²⁴.

Finally, in an embodiment, two segments of the polypeptide chain are not visible in the electron density. The gaps include residues between K¹⁸² and D¹⁹⁰ and between F¹⁴⁶ and N¹⁵⁰.

In an embodiment, there is provided a method of preparing the crystal of murine glutaminyl cyclase as described herein, which comprises the steps of:

-   -   (a) providing a solution of murine glutaminyl cyclase,         optionally in the presence of a known glutaminyl cyclase         inhibitor, in a suitable buffer such as 15 mM Bis-Tris pH6.8/100         mM NaCl buffer;     -   (b) mixing the solution with a crystallization solution         comprising 0.1M sodium acetate pH 5.3, 0.2M ammonium sulphate         and 12% (w/v) 2000 MME-PEG; and     -   (c) incubating the mixture under conditions to promote hanging         drop vapor diffusion for a time sufficient to produce the         crystal of murine glutaminyl cyclase.

In an embodiment, there is provided a method of preparing the co-crystal of human glutaminyl cyclase bound to a binding ligand, such as a glutaminyl cyclase inhibitor, which comprises the steps of:

-   -   (a) providing a solution of murine glutaminyl cyclase in the         presence of a known glutaminyl cyclase inhibitor, in a suitable         buffer such as 15 mM Bis-Tris pH6.8/100 mM NaCl buffer;     -   (b) mixing the solution with a crystallization solution         comprising 0.1M sodium acetate pH 5.3, 0.2M ammonium sulphate         and 12% (w/v) 2000 MME-PEG; and     -   (c) incubating the mixture under conditions to promote hanging         drop vapor diffusion for a time sufficient to produce the         co-crystal of murine glutaminyl cyclase bound to a binding         ligand, such as a glutaminyl cyclase inhibitor.

In an embodiment, there is provided a crystal or co-crystal comprising murine glutaminyl cyclase obtainable by the crystallisation method as defined herein.

Crystallization of murine glutaminyl cyclase to produce co-crystals can be carried out as described below and in Example 2. As described, purified murine glutaminyl cyclase is concentrated to 10 mg/mL in the presence of a suitable buffer, such as 15 mM Bis-Tris pH 6.8/100 mM NaCl buffer and the ligand, Inhibitor A, Inhibitor B or Inhibitor C, is added to a final concentration of 1 mM. Macroscopic rod/needle crystal forms are grown by hanging drop vapor diffusion at 21° C. by mixing an equal volume of protein solution with a crystallization solution comprising 0.1M sodium acetate pH 5.3, 0.2M ammonium sulphate and 12% (w/v) 2000 MME-PEG. Crystals typically appeared within 1 week after the experiment was initiated and examples of such crystals obtained following this protocol are shown in FIG. 14. It will be appreciated that to produce apo-crystals, the ligand is omitted in the above protocol.

The murine glutaminyl cyclase crystals may be frozen prior to data collection. The crystals can be cryo-protected with for example, either (a) 20-30% saturated glucose present in the crystallization setup, (b) ethanol added to 15-20%, (c) ethylene glycol added to 10-20% and PEG10,000 brought up to 25%, or (d) glycerol added to 20%. In an embodiment, the crystals can be cryo-protected with the addition of glycerol added to 20% (v/v). The crystals can be either briefly immersed in the cryo-protectant or soaked in the cryo-protectant for periods as long as a day. Freezing can be accomplished by immersing the crystal in a bath of liquid nitrogen or by placing the crystal in a stream of nitrogen gas at −180° C.

As described in Example 2, the crystal structure of murine glutaminyl cyclase in complex with glutaminyl cyclase inhibitors Inhibitor A, Inhibitor B and Inhibitor C was obtained. A summary of the crystal's attributes for murine glutaminyl cyclase bound to glutaminyl cyclase inhibitors Inhibitor A, Inhibitor B and Inhibitor C are listed in Table 4. The three dimensional structure coordinates for space groups P212121 for murine glutaminyl cyclase bound to glutaminyl cyclase inhibitor Inhibitor A is shown in FIG. 2. The three dimensional structure coordinates for space groups P212121 for murine glutaminyl cyclase bound to glutaminyl cyclase inhibitor Inhibitor B is shown in FIG. 3. The three dimensional structure coordinates for space groups P212121 for murine glutaminyl cyclase bound to glutaminyl cyclase inhibitor Inhibitor C is shown in FIG. 4. Ribbon and overlay diagrams of murine glutaminyl cyclase bound to glutaminyl cyclase inhibitors Inhibitor A, Inhibitor B and Inhibitor C based upon the coordinates for space groups P212121 are shown in FIGS. 15 to 19. In particular, it was found that the protein comprised a globular α/β hydrolase fold. A central β-sheet was formed with six β-strands all in parallel fashion but not the second. This β-sheet was observed to be surrounded by α-helices in a sandwich manner with two helices in one side and six more α-helices in the opposite face. The protein's structure was completed with a rather large amount of random coiled loops which appeared to build the active site of the enzyme. This active site accommodates a zinc ion which is coordinated by three protein residues D¹⁶⁰, E²⁰³ and H³³¹ and an inhibitor molecule. Thus, in an embodiment, the crystal can comprise a binding pocket provided by residues D160, E203 and H331 of SEQ ID NO: 13 according to the coordinates of FIGS. 2 to 4.

Furthermore, the protein demonstrated the presence of a disulfide bridge between residues C¹⁴⁰ and C¹⁶⁵. Thus, in an embodiment, the crystal can comprise a disulfide bridge between residues C140 and C165 of SEQ ID NO: 13. As discussed hereinbefore, the presence of such a disulfide bridge has not previously been reported during crystallisation studies with QC (Wang et al, Huang et al).

The present disclosure also provides a machine-readable data storage media having data storage material encoded with machine-readable data that comprises structure coordinates for glutaminyl cyclase. The present invendisclosure also provides a machine readable data storage media having data storage material encoded with machine readable data, which, when read by an appropriate machine, can display a three dimensional representation of a structure of glutaminyl cyclase.

All or a portion of glutaminyl cyclase coordinate data shown in FIGS. 1 to 4, when used in conjunction with a computer programmed with software to translate those coordinates into the three-dimensional structure of glutaminyl cyclase may be used for a variety of purposes, especially for purposes relating to drug discovery. Software for generating three-dimensional graphical representations are known and commercially available. The ready use of the coordinate data requires that it be stored in a computer-readable format. Thus, in accordance with the present disclosure, data capable of being displayed as the three-dimensional structure of glutaminyl cyclase or portions thereof or their structurally similar variants may be stored in a machine-readable storage medium, which is capable of displaying a graphical three-dimensional representation of the structure.

In an embodiment, there is provided a machine-readable data storage medium, comprising a data storage material encoded with machine readable data which, when used by a machine programmed with instructions for using said data, displays a graphical three-dimensional representation comprising glutaminyl cyclase or variant thereof.

Optionally, a computer system can be provided in combination with the machine-readable data storage medium provided herein. In an embodiment, the computer system can comprise a working memory for storing instructions for processing the machine-readable data; a processing unit coupled to the working memory and to the machine-readable data storage medium, for processing the machine-readable data into the three-dimensional representation; and an output hardware coupled to the processing unit, for receiving the three-dimensional representation.

The three-dimensional crystal structure of the present disclosure may be used to identify glutaminyl cyclase binding sites, be used as a molecular replacement model to solve the structure of unknown crystallized proteins, to design mutants having desirable binding properties, and ultimately, to design, characterize, identify entities capable of binding and inhibiting glutaminyl cyclase and other structurally similar proteins as well as other uses that would be recognized by one of ordinary skill in the art. Such entities may be chemical entities or proteins. The term “chemical entity”, as used herein, refers to chemical compounds, complexes of at least two chemical compounds, and fragments of such compounds.

The glutaminyl cyclase structural coordinates provided herein are useful for screening and identifying drugs that inhibit glutaminyl cyclase and other structurally similar proteins. For example, the structure encoded by the data may be computationally evaluated for its ability to associate with putative substrates or ligands. Such compounds that associate with glutaminyl cyclase may inhibit glutaminyl cyclase activity, and are potential drug candidates. Additionally or alternatively, the structure encoded by the data may be displayed in a graphical three-dimensional representation on a computer screen. This allows visual inspection of the structure, as well as visual inspection of the structure's association with the compounds.

Thus, in an embodiment, there is provided a method of identifying an inhibitor of human glutaminyl cyclase which comprises the following steps:

-   -   (a) generating a 3-dimensional model of human glutaminyl cyclase         using the structural coordinates described in FIG. 1;     -   (b) analysing the binding pocket provided by residues E202, D159         and H330 of SEQ ID NO: 1 according to the coordinates of FIG. 1;     -   (c) performing computer modeling analysis to identify an         inhibitor compound which may associate with the binding pocket         of human glutaminyl cyclase.

Furthermore, in an embodiment, there is provided a method of identifying an inhibitor of murine glutaminyl cyclase which comprises the following steps:

-   -   (a) generating a 3-dimensional model of murine glutaminyl         cyclase using the structural coordinates described in any of         FIGS. 2 to 4;     -   (b) analysing the binding pocket provided by residues D160, E203         and H331 of SEQ ID NO: 13 according to the coordinates of FIGS.         2 to 4;     -   (c) performing computer modeling analysis to identify an         inhibitor compound which may associate with the binding pocket         of murine glutaminyl cyclase.

In an embodiment, a method is also provided for evaluating the potential of an entity to associate with glutaminyl cyclase or variant thereof by using all or a portion of the structure coordinates provided in FIGS. 1 to 4 or functional equivalents thereof. In an embodiment, a method is also provided for evaluating the potential of an entity to associate with glutaminyl cyclase or variant thereof by using structure coordinates similar to all or a portion of the structure coordinates provided in FIGS. 1 to 4 or functional equivalents thereof.

In an embodiment, the method can additionally comprise the step of synthesizing the inhibitor compound and contacting the compound with the binding pocket of glutaminyl cyclase to determine the ability of the compound to inhibit glutaminyl cyclase.

In an embodiment, the step of performing computer modeling analysis to identify said inhibitor compound can comprise identifying said compound from a library of compounds.

In an embodiment, the step of performing computer modeling analysis to identify said inhibitor compound can comprise identifying said compound in a database.

In an embodiment, the step of performing computer modeling analysis to identify said inhibitor compound can comprise designing the compound from a known glutaminyl cyclase inhibitor.

With the structures provided herein, the present disclosure permits the use of molecular design techniques to identify, select or design potential inhibitors of glutaminyl cyclase based on the structure of glutaminyl cyclase. Such a predictive model can be valuable in light of the high costs associated with the preparation and testing of the many diverse compounds that may possibly bind to glutaminyl cyclase.

According to the present disclosure, a potential glutaminyl cyclase inhibitor may be evaluated for its ability to bind glutaminyl cyclase prior to its actual synthesis and testing. If a proposed entity is predicted to have insufficient interaction or association with the binding pocket, preparation and testing of the entity can be obviated. However, if the computer modeling indicates a strong interaction, the entity may then be obtained and tested for its ability to bind.

A potential inhibitor of glutaminyl cyclase may be computationally evaluated using a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with glutaminyl cyclase.

One skilled in the art may use one of several methods to screen entities (whether chemical or protein) for their ability to associate with glutaminyl cyclase. This process may begin by visual inspection of, for example, glutaminyl cyclase on a computer screen based on the glutaminyl cyclase structure coordinates in FIGS. 1 to 4 or other coordinates which define a similar shape generated from the machine-readable storage medium. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within that binding pocket as defined above. Docking may be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM and AMBER.

Specialized computer programs may also assist in the process of selecting entities. These include: GRID (Goodford, “A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules”, J. Med. Chem., 28, pp. 849-857 (1985)). GRID is available from Oxford University, Oxford, UK; MCSS (Miranker et al., “Functionality. Maps of Binding Sites: A Multiple Copy Simultaneous Search Method.” Proteins: Structure, Function and Genetics, 11, pp. 29-34 (1991)). MCSS is available from Molecular Simulations, San Diego, Calif.; AUTODOCK (Goodsell et al., “Automated Docking of Substrates to Proteins by Simulated Annealing”, Proteins: Structure, Function, and Genetics, 8, pp. 195-202 (1990)). AUTODOCK is available from Scripps Research Institute, La Jolla, Calif.; & DOCK (Kuntz et al., “A Geometric Approach to Macromolecule-Ligand Interactions”, J. Mol. Biol., 161, pp. 269-288 (1982)). DOCK is available from University of California, San Francisco, Calif.

Once suitable entities have been selected, they can be designed or assembled. Assembly may be preceded by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of glutaminyl cyclase. This may then be followed by manual model building using software such as MOE, QUANTA or Sybyl (Tripos Associates, St. Louis, Mo.).

Useful programs to aid one of skill in the art in connecting the individual chemical entities or fragments include: CAVEAT (Bartlett et al., “CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules”, in “Molecular Recognition in Chemical and Biological Problems”, Special Pub., Royal Chem. Soc., 78, pp. 182-196 (1989); Lauri and Bartlett, “CAVEAT: a Program to Facilitate the Design of Organic Molecules”, J. Comput. Aided Mol. Des., 8, pp. 51-66 (1994)). CAVEAT is available from the University of California, Berkeley, Calif.; 3D Database systems such as ISIS (MDL Information Systems, San Leandro, Calif.). This area is reviewed in Martin, “3D. Database Searching in Drug Design”, J. Med. Chem., 35, pp. 2.145-2154 (1992); HOOK (Eisen et al., “HOOK: A Program for Finding Novel Molecular Architectures that Satisfy the Chemical and Steric Requirements of a Macromolecule Binding Site”, Proteins: Struct., Funct., Genet., 19, pp. 199-221 (1994). HOOK is available from Molecular Simulations, San Diego, Calif.

Instead of proceeding to build an inhibitor of glutaminyl cyclase in a step-wise fashion one fragment or entity at a time as described above, inhibitory or other glutaminyl cyclase binding compounds may be designed as a whole or “de novo” using either an empty binding site or optionally including some portion(s) of a known inhibitor(s). There are many de novo ligand design methods including: LUDI (Bohm, “The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors”, J. Comp. Aid. Molec. Design, 6, pp. 61-78 (1992)). LUDI is available from Molecular Simulations Incorporated, San Diego, Calif.; LEGEND (Nishibata et al., Tetrahedron, 47, p. 8985 (1991)). LEGEND is available from Molecular Simulations Incorporated, San Diego, Calif.; LEAPFROG (available from Tripos Associates, St. Louis, Mo.); & SPROUT (Gillet et al., “SPROUT: A Program for Structure Generation)”, J. Comput. Aided Mol. Design, 7, pp. 127-153 (1993)). SPROUT is available from the University of Leeds, UK.

Other molecular modeling techniques may also be employed in accordance with the present disclosure (See, for example, Cohen et al., “Molecular Modeling Software and Methods for Medicinal Chemistry, J. Med. Chem., 33, pp. 883-894 (1990); See also, Navia and Murcko, “The Use of Structural Information in Drug Design”, Current Opinions in Structural Biology, 2, pp. 202-210 (1992); Balbes et al., “A Perspective of Modern Methods in Computer-Aided Drug Design”, in Reviews in Computational Chemistry, Vol. 5, Lipkowitz and Boyd, Eds., VCH, New York, pp. 337-380 (1994); See also, Guida, “Software For Structure-Based Drug Design”, Curr. Opin. Struct. Biology, 4, pp. 777-781 (1994)).

Once an entity has been designed or selected, for example, by the above methods, the efficiency with which that entity may bind to glutaminyl cyclase may be tested and optimized by computational evaluation. For example, an effective glutaminyl cyclase inhibitor preferably demonstrates a relatively small difference in energy between its bound and free states (i.e., a small deformation energy of binding). Thus, the most efficient glutaminyl cyclase inhibitors should preferably be designed with deformation energy of binding of not greater than about 10 kcal/mole, more preferably, not greater than 7 kcal/mole. Glutaminyl cyclase inhibitors may interact with the protein in more than one of multiple conformations that are similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the free entity and the average energy of the conformations observed when the inhibitor binds to the protein.

An entity designed or selected as binding to glutaminyl cyclase may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target enzyme and with the surrounding water molecules. Such non-complementary electrostatic interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions.

Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interactions. Examples of programs designed for such uses include: Gaussian 94, revision C (Frisch, Gaussian, Inc., Pittsburgh, Pa. 1995); AMBER, version 4.1 (Kollman, University of California at San Francisco, 1995); QUANTA/CHARMM (Molecular Simulations, Inc., San Diego, Calif. 1995); Insight II/Discover (Molecular Simulations, Inc., San Diego, Calif. 1995); DelPhi (Molecular Simulations, Inc., San Diego, Calif. 1995); and AMSOL (Quantum Chemistry Program Exchange, Indiana University). These programs may be implemented, for instance, using a Silicon Graphics workstation such as an Indigo.sup.2 with “IMPACT” graphics. Other hardware systems and software packages will be known to those skilled in the art.

Another approach provided by an embodiment of the present disclosure, is the computational screening of small molecule databases for chemical entities or compounds that can bind in whole, or in part, to glutaminyl cyclase. In this screening, the quality of fit of such entities to the binding site may be judged either by shape complementarities or by estimated interaction energy (Meng et al., J. Comp. Chem., 13, 505-524 (1992)).

In an embodiment, there is provided a compound that associates with glutaminyl cyclase produced or identified by various methods as described hereinbefore.

The structure coordinates set forth in FIGS. 1 to 4 can also be used to aid in obtaining structural information about another crystallized molecule or molecular complex. This may be achieved by any of a number of well-known techniques, including molecular replacement.

For example, a method is also provided for utilizing molecular replacement to obtain structural information about a protein whose structure is unknown comprising the steps of: generating an X-ray diffraction pattern of a crystal of the protein whose structure is unknown; generating a three-dimensional electron density map of the protein whose structure is unknown from the X-ray diffraction pattern by using at least a portion of the structure coordinates set forth in FIGS. 1 to 4 as a molecular replacement model.

By using molecular replacement, all or part of the structure coordinates of glutaminyl cyclase provided by the present disclosure (and set forth in FIGS. 1 to 4) can be used to determine the structure of another crystallized molecule or molecular complex more quickly and efficiently than attempting an ab initio structure determination. One particular use includes use with other structurally similar proteins. Molecular replacement provides an accurate estimation of the phases for an unknown structure. Phases are a factor in equations used to solve crystal structures that cannot be determined directly. Obtaining accurate values for the phases, by methods other than molecular replacement, is a time-consuming process that involves iterative cycles of approximations and refinements and greatly hinders the solution of crystal structures. However, when the crystal structure of a protein containing at least a homologous portion has been solved, the phases from the known structure provide a satisfactory estimate of the phases for the unknown structure.

Thus, this method involves generating a preliminary model of a molecule or molecular complex whose structure coordinates are unknown, by orienting and positioning the relevant portion of glutaminyl cyclase according to FIGS. 1 to 4 within the unit cell of the crystal of the unknown molecule or molecular complex so as best to account for the observed X-ray diffraction pattern of the crystal of the molecule or molecular complex whose structure is unknown. Phases can then be calculated from this model and combined with the observed X-ray diffraction pattern amplitudes to generate an electron density map of the structure whose coordinates are unknown. This, in turn, can be subjected to any well-known model building and structure refinement techniques to provide a final, accurate structure of the unknown crystallized molecule or molecular complex (Lattman, “Use of the Rotation and Translation Functions”, in Meth. Enzymol., 115, pp. 55-77 (1985); Rossmann, ed., “The Molecular Replacement Method”, Int. Sci. Rev. Ser., No. 13, Gordon & Breach, New York (1972)).

The structure of any portion of any crystallized molecule or molecular complex that is sufficiently homologous to any portion of glutaminyl cyclase can be resolved by this method.

In one embodiment, the method of molecular replacement is utilized to obtain structural information about the present disclosure and any other glutaminyl cyclase-like molecule.

The structure coordinates of glutaminyl cyclase as provided by the present disclosure are useful in solving the structure of glutaminyl cyclase variants that have amino acid substitutions, additions and/or deletions (referred to collectively as “glutaminyl cyclase mutants”, as compared to naturally occurring glutaminyl cyclase). These glutaminyl cyclase mutants may optionally be crystallized in co-complex with a ligand, such as an inhibitor or substrate analogue. The crystal structures of a series of such complexes may then be solved by molecular replacement and compared with that of glutaminyl cyclase. Potential sites for modification within the various binding sites of the enzyme may thus be identified. This information provides an additional tool for determining the most efficient binding interactions such as, for example, increased hydrophobic interactions, between glutaminyl cyclase and a ligand. It is noted that the ligand may be the protein's natural ligand or may be a potential agonist or antagonist of a protein.

In an embodiment, there is provided a method of designing, selecting and/or optimising a chemical entity that binds to all or part of the binding pocket or human or murine glutaminyl cyclase according to any one of FIGS. 1 to 4 comprising the steps of:

-   -   (a) providing the structural coordinates of said binding pocket         or human or murine glutaminyl cyclase on a computer comprising         the means for generating 3-dimensional structural information         from said structural coordinates; and     -   (b) designing selecting and/or optimising said chemical entity         by performing a fitting operation between said chemical entity         and said 3-dimensional structural information of all or part of         said binding pocket or human or murine glutaminyl cyclase.

In an embodiment, there is provided a method for evaluating the ability of a chemical entity to associate with all or part of the binding pocket or human or murine glutaminyl cyclase according to any one of FIGS. 1 to 4 comprising the steps of:

-   -   (a) providing the structural coordinates of said binding pocket         or human or murine glutaminyl cyclase on a computer comprising         the means for generating 3-dimensional structural information         from said structural coordinates;     -   (b) employing computational means to perform a fitting operation         between the chemical entity and all or part of the binding         pocket or human or murine glutaminyl cyclase; and     -   (c) analyzing the results of said fitting operation to         quantitate the association between the chemical entity and all         or part of the binding pocket or human or murine glutaminyl         cyclase.

In an embodiment, the method can further comprise generating a 3-dimensional graphical representation of all or part of the binding pocket or human or murine glutaminyl cyclase prior to step (b).

In an embodiment, there is provided a method of using a computer for evaluating the ability of a chemical entity to associate with all or part of the binding pocket or human or murine glutaminyl cyclase according to any one of FIGS. 1 to 4, wherein said computer comprises a machine-readable data storage medium comprising a data storage material encoded with said structural coordinates defining said binding pocket or human or murine glutaminyl cyclase and means for generating a three-dimensional graphical representation of the binding pocket or human or murine glutaminyl cyclase, and wherein said method comprises the steps of:

-   -   (a) positioning a first chemical entity within all or part of         said binding pocket or human or murine glutaminyl cyclase using         a graphical 3-dimensional representation of the structure of the         chemical entity and the binding pocket or human or murine         glutaminyl cyclase;     -   (b) performing a fitting operation between said chemical entity         and said binding pocket or human or murine glutaminyl cyclase by         employing computational means; and     -   (c) analyzing the results of said fitting operation to         quantitate the association between said chemical entity and all         or part of the binding pocket or human or murine glutaminyl         cyclase.

In an embodiment, the method can further comprise the steps of:

-   -   (d) repeating steps (a) through (c) with a second chemical         entity; and     -   (e) selecting at least one of said first or second chemical         entity that associates with said all or part of said binding         pocket or human or murine glutaminyl cyclase based on said         quantitated association of said first or second chemical entity.

In an embodiment, there is provided a method for identifying an agonist or antagonist of the human or murine glutaminyl cyclase according to any one of FIGS. 1 to 4, comprising the steps of:

-   -   (a) using a 3-dimensional structure of the binding pocket or         human or murine glutaminyl cyclase to design or select a         chemical entity;     -   (b) contacting the chemical entity with the human or murine         glutaminyl cyclase;     -   (c) monitoring the catalytic activity of the human or murine         glutaminyl cyclase; and     -   (d) classifying the chemical entity as an agonist or antagonist         based on the effect of the chemical entity on the catalytic         activity of human or murine glutaminyl cyclase.

In an embodiment, there is provided a method of designing a compound or complex that associates with all or part of the binding pocket or human or murine glutaminyl cyclase according to any one of FIGS. 1 to 4, comprising the steps of:

-   -   (a) providing the structural coordinates of said binding pocket         or human or murine glutaminyl cyclase on a computer comprising         the means for generating 3-dimensional structural information         from said structural coordinates; and     -   (b) using the computer to perform a fitting operation to         associate a first chemical entity with all or part of the         binding pocket;     -   (c) performing a fitting operation to associate at least a         second chemical entity with all or part of the binding pocket;     -   (d) quantifying the association between the first and second         chemical entity and all or part of the binding pocket;     -   (e) optionally repeating steps (b) to (d) with another first and         second chemical entity, selecting a first and a second chemical         entity based on said quantified association of all of said first         and second chemical entity;     -   (f) optionally, visually inspecting the relationship of the         first and second chemical entity to each other in relation to         the binding pocket on a computer screen using the 3-dimensional         graphical representation of the binding pocket and said first         and second chemical entity; and     -   (g) assembling the first and second chemical entity into a         compound or complex that associates with all or part of said         binding pocket by model building.

Any, all or a portion of the complexes referred to above may be studied using well-known X-ray diffraction techniques and may be refined versus 1.5-3.5 Å resolution X-ray data to an R value of about 0.22 or less using computer software, such as X-PLOR (Brunger et al., X-PLOR, Version 3.1, A system for X-ray crystallography and NMR, Yale University, (1992)), CNS (Brunger et al., Crystallography & NMR System (CNS), A new software suite for macromolecular structure determination, Acta Cryst. D54: 905-921 (1998)), TNT (Tronrud et al., An efficient general-Purpose least-squares refinement program for macromolecular structures, Acta Cryst. A43, 489-501 (1987)), Buster (Bricogne, The Bayesian Statistical Viewpoint on Structure Determination: Basic Concepts and Examples”, in Methods in Enzymology, 276A, 361-423. Carter & Sweet, eds. (1997)) and Refmac (Murshudov et al., Refinement of macromolecular structures by the maximum-likelihood method, Acta Cryst D53:240-255 (1997)) (See, e.g., Blundell & Johnson, supra; Meth. Enzymol., Vol. 114 & 115, Wyckoff et al., eds., Academic Press (1985)). This information may thus be used to optimize known glutaminyl cyclase inhibitors, and more importantly, to design new glutaminyl cyclase inhibitors.

The structure coordinates described herein may also be used to derive the dihedral angles, phi and psi, that define the conformation of the amino acids in the protein backbone. As will be understood by those skilled in the art, the phi angle refers to the rotation around the bond between the alpha-carbon and the nitrogen, and the psi angle refers to the rotation around the bond between the carbonyl carbon and the alpha-carbon. The subscript “n” identifies the amino acid whose conformation is being described (for a general reference, See Blundell and Johnson, Protein Crystallography, Academic Press, London, 1976).

Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

Preparation of 1-(3-(1H-Imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)thiourea (Inhibitor A)

4.0 mmol of 3,4-dimethoxyphenyl isothiocyanate and 4.0 mmol of 3-(1H-imidazol-1-yl)alkyl-1-amine were dissolved in 10 mL of absolute ethanol. After stirring for 2 h under reflux, the solvent was evaporated and the resulting solid was recrystallized from ethanol. Yield: 0.66 g (51.3%); mp: 160.0-161.0° C.

¹H NMR δ 1.8-2.0 (m, 2H), 3.4-3.5 (m, 2H), 3.75 (s, 6H), 3.9-4.0 (m, 2H), 6.7-6.8 (m, 1H), 6.9 (br m, 2H), 6.95 (s, 1H), 7.15 (s, 1H), 7.55 (br s, 1H), 7.6 (s, 1H), 9.3 (s, 1H); MS m/z 321.2 (M+H), 253.3 (M-C₃H₃N₂.)

Preparation of 2-Cyano(3,4,5-trimethoxyphenyl)-3-(3-(4-methyl-1H-imidazol-1-yl)propyl)-guanidine (Inhibitor B) Intermediate 1: 4-Methyl-1-trityl-1H-imidazole (D1)

4-Methyl-1H-imidazole (36.53 mmol, 1 eq) of was dissolved in 120 mL of dimethylformamide, triethylamine (73.06 mmol, 2 eq.) and chlorotriphenylmethane (40.1 mmol, 1.1 eq) where added. The mixture was stirred for 3.5 h. The precipitate filtered off and was washed by means of ice-cooled dimethylformamide (2×50 mL) and water (2×50 mL). After removal of the solvent the remaining product was dried over P₄O₁₀.

Yield: 10.65 g (98.2%). The product was used without further purification.

Intermediate 2: 1-Trityl-3-[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]-1,4-dimethyl-1H-imidazol-3-ium bromide (D2)

4-Methyl-1-trityl-1H-imidazole (i.e 32.85 mmol, 1 eq., which may be prepared as described in D1) was suspended in acetonitrile (10 mL) and 2-(3-bromopropyl)isoindoline-1,3-dione (32.85 mmol, 1 eq.) was added. The mixture was kept under reflux over night. The organic solvent was removed.

Purification was done by flash-chromatography using silica gel and a CHCl₃/MeOH-gradient.

Yield: 10.65 g (63.44%).

Intermediate 3: 2-(3-(5-Methyl-1H-imidazol-1-yl)propyl)isoindoline-1,3-dione (D3)

1-Trityl-3-[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]-1,4-dimethyl-1H-imidazol-3-ium bromide (i.e. 7.86 mmol, which may be prepared as described in D2) was dissolved in a stirred solution containing methanol (20 mL) and trifluoracetic acid (4 mL). The mixture was kept under reflux over night. The solvent was then removed by means of reduced pressure and the remaining oil was purified by flash-chromatography using silica gel and a CHCl₃/MeOH-gradient.

Yield: 2.05 g (97.0%).

Intermediate 4: 3-(5-Methyl-1H-imidazol-1-yl)propan-1-amine (D4)

2-(3-(5-Methyl-1H-imidazol-1-yl)propyl)isoindoline-1,3-dione (i.e. 8.92 mmol, 1 eq., which may be prepared as described in D3) and hydrazine monohydrate (17.84 mmol, 2 eq.) were dissolved in dry EtOH (50 mL). The mixture was kept under reflux over night, then mixture was concentrated down to a volume of 25 mL. Hydrochloric acid (conc., 55 mL) was then added and the mixture was heated to 50° C. and kept at this temperature for 30 min. The formed precipitate was then filtered. The filtrate was cooled to 0° C. and solid NaOH was added until a final pH-value of 10-12 was reached. The aqueous solution was extracted by means of CHCl₃ (3×50 mL). The combined organic layers were dried over Na₂SO₄, filtered and the solvent was removed. The product was purified by means of flash-chromatography using silica gel and a CHCl₃/MeOH-gradient.

Yield: 0.74 g (60%), viscous oil

Yield over all steps: 36.3%

¹H-NMR (CDCl₃, 499.78 MHz): δ 1.79-1.847 (m, 2H); 2.179 (s, 3H); 2.694-2.721 (m, 2H); 3.891-3.920 (m, 2H); 6.731 (s, H); 7.240 (s, solv.); 7.380 (s; H); ESI-MS m/z: 140.3 (M+H)⁺, 279.4 (2M+H)⁺; HPLC (λ=214 nm) rt: dead time (100%)

Intermediate 5: 2-(3-(4/5-methyl-1H-imidazol-1-yl)propyl)isoindoline-1,3-dione (D5)

4-Methyl-1H-imidazole (36.53 mmol, 1 eq) and sodium hydride (60% in mineral oil, 36.53 mmol, 1.0 eq.) were dissolved in 80 mL of dimethylformamide. The mixture was stirred at room temperature for 2 h until the formation of hydrogen gas deceased. 2-(3-Bromopropyl)isoindoline-1,3-dione (34.70 mmol, 0.95 eq.) was added and the mixture was stirred at 90° C. overnight. The solvent was removed and the remaining residue was purified by means of flash-chromatography using silica gel and a CHCl₃/MeOH-gradient.

Yield: 6.1 g (62.0%) of a mixture of 2-(3-(4-methyl-1H-imidazol-1-yl)propyl)isoindoline-1,3-dione and 2-(3-(5-methyl-1H-imidazol-1-yl)propyl)isoindoline-1,3-dione

Intermediate 6: 2-(3-(4-Methyl-1H-imidazol-1-yl)propyl)isoindoline-1,3-dione (D6)

A mixture consisting of 2-(3-(4-methyl-1H-imidazol-1-yl)propyl)isoindoline-1,3-dione and 2-(3-(5-methyl-1H-imidazol-1-yl)propyl)isoindoline-1,3-dione (22.65 mmol, 1 eq., which may be prepared as described in D5) and trityl chloride (13.6 mmol, 0.6 eq.) were dissolved in 40 mL of dichloromethane and kept at a temperature of 0° C. for 10 min and 1.5 h at room temperature. The solvent was removed and the remaining solid was purified by means of flash-chromatography using silica gel and a CHCl₃/MeOH-gradient.

Yield: 0.92 g (15.1%)

Intermediate 7: 3-(4-Methyl-1H-imidazol-1-yl)propan-1-amine (D7)

2-(3-(4-Methyl-1H-imidazol-1-yl)propyl)isoindoline-1,3-dione (3.42 mmol, 1 eq., which may be prepared as described in D6) and hydrazine monohydrate (6.84 mmol, 2 eq.) were dissolved in 20 mL of ethanol and the mixture was stirred for 12 h under reflux. The mixture was kept under reflux over night, then the mixture was concentrated down to a volume of 25 mL. Hydrochloric acid (conc., 55 mL) was then added and the mixture was heated to 50° C. and kept at this temperature for 30 min. The formed precipitate was then filtered. The filtrate was cooled to 0° C. and solid NaOH was added until a final pH-value of 10-12 was reached. The aqueous solution was extracted by means of CHCl₃ (3×50 mL). The combined organic layers were dried over Na₂SO₄, filtered and the solvent was removed. The product was purified by means of flash-chromatography using silica gel and a CHCl₃/MeOH-gradient containing aqueous ammonia (2% v/v).

Yield: 0.31 g (65.1%).

¹H-NMR (CDCl₃, 499.78 MHz): δ 1.819-1.874 (m, 2H); 2.188 (s, 3H); 2.699-2.712 (m, 2H); 3.910-3.948 (m, 2H); 6.594 (s, H); 7.240 (s, solv.); 7.328 (s; H); ESI-MS m/z: 140.3 (M+H)⁺, 279.4 (2M+H)⁺; HPLC (λ=214 nm) rt: dead time (100%)

Inhibitor B: 2-Cyano(3,4,5-trimethoxyphenyl)-3-(3-(4-methyl-1H-imidazol-1-yl)propyl)-guanidine

Inhibitor B was synthesized from 5-isothiocyanato-1,2,3-trimethoxybenzene (0.162 g, 0.72 mmol) and 3-(4-methyl-1H-imidazol-1-yl)propan-1-amine (0.10 g, 0.72 mmol, which may be prepared as described in D7).

Yield: 0.110 g (41.0%). ¹H NMR (CDCl₃): δ 1.95-2.01 (m, 2H); 2.14 (s, 3H); 3.22-3.27 (m, 2H); 3.82 (s, 9H); 3.86-3.88 (m, 2H); 5.03 (br s, H); 6.41 (s, 2H); 6.56 (s, H); 7.28 (s, H); 7.46 (s, H). MS m/z 373.3 (M+H)⁺; HPLC (λ=214 nm, [C]): rt 8.64 min (100%)

Preparation of 2-Cyano(mesityl)-3-(3-(4-methyl-1H-imidazol-1-yl)propyl)guanidine (Inhibitor C)

Inhibitor C was synthesized from 2-isothiocyanato-1,3,5-trimethylbenzene (0.106 g, 0.60 mmol) and 3-(4-methyl-1H-imidazol-1-yl)propan-1-amine (0.084 g, 0.60 mmol, which may be prepared as described in D7).

Yield: 0.088 g (45.2%). ¹H NMR (DMSO-d₆): δ 1.80-1.84 (m, 2H); 2.04 (s, 3H); 2.09 (s, 6H); 2.23 (s, 3H); 3.04-3.06 (m, 2H); 3.80-3.84 (m, 2H); 6.59 (br s, H); 6.80 (s, H); 6.91 (s, 2H); 7.42 (s, H); 8.42 (s, H). MS m/z 325.2 (M+H)⁺; HPLC (λ=214 nm, [C]): rt 10.05 min (98.6%)

Example 1 Isolation, Characterisation and Crystallisation of Human Glutaminyl Cyclase

(A) Isolation of Human Glutaminyl Cyclase

Cloning Procedures

The hQC cDNA was inserted into the yeast expression vector ρPICZαB (Invitrogen, Germany) via the PstI and NotI restriction sites using the primers:

(sense)(P1) (SEQ ID NO: 2) 5′-ATATATCTGCAGCG CAT CAC CAT CAC CAT CAC GAG GAG AAG AAT TAC CAC C-3; and (antisense)(P2) (SEQ ID NO: 3) 5′-ATATATGCGGCCGC TTA CAA ATG AAG ATA TTC C-3′.

The disulfide bond-lacking mutant C139A/C164A was obtained via a two-step PCR-mediated site-directed mutagenesis according to standard PCR techniques using the primer pairs:

C139A (sense) (SEQ ID NO: 4) 5′-CAT TTG GTC CTC GCC GCC CAC TAT GAC TCC AAG- 3′; C139A (antisense) (SEQ ID NO: 5) 5′-CTT GGA GTC ATA GTG GGC GGC GAG GAC CAA ATG- 3′; C164A (sense) (SEQ ID NO: 6)  5′-GAT TCA GCC GTG CCA GCT GCA ATG ATG TTG GAA  C-3′; and C164A (antisense) (SEQ ID NO: 7)  5′-G TTC CAA CAT CAT TGC AGC TGG CAC GGC TGA ATC- 3′ followed by digestion of the parent DNA using  Dpnl (quik-change II site-directed mutagenesis kit, Stratagene, USA). Transformation of Pichia pastoris and Mini-Scale Expression

Plasmid DNA was amplified in the Escherichia coli strain DH5a and purified according to the recommendations of the manufacturer (Qiagen, Germany). 20-30 μg DNA were linearised with PmeI, precipitated, and dissolved in deionised water. 1-5 μg DNA was used for transformation of competent yeast cells (P. pastoris strain X33 [AOX1, AOX2]) by electroporation according to the manufacturer's instructions (Bio-Rad, Germany). Yeast was grown, transformed, and analysed according to the manufacturer's instructions (Invitrogen, Germany). Briefly, selection was carried out on YPDS plates containing 150 μg/ml zeocin. To test yeast clones upon expression, recombinants were grown for 24 h in 10 ml conical tubes containing 2 ml of BMGY. Afterwards, the cells were centrifuged and resuspended in 2 ml of BMMY containing 0.5% methanol. This concentration was maintained by addition of methanol every 24 h. QC activity was determined in the supernatant after 72 h. The expression of the C139A/C164A mutant was confirmed by sequence analysis of the hQC gene inserted into the yeast genome applying the primer 5′-CTG GAG TGA CAA ATC TGG C-3′ (SEQ ID NO: 8). Therefore genomic DNA was prepared according to standard molecular biological techniques followed by the amplification of the target gene via PCR using primers (P1; SEQ ID NO: 2) and (P2; SEQ ID NO: 3). Clones displaying highest QC activity were chosen for fermentation and further experiments.

Large Scale Expression and Purification

Fermentation of hQC and the C139A/C164A mutant was carried out in a 5 L reactor (Biostad B; Braun Biotech, Germany), essentially as described elsewhere (Schilling, S., Hoffmann, T., Rosche, F., Manhart, S., Wasternack, C., and Demuth, H. U. [2002] Biochemistry 41, 10849-10857). Briefly, fermentation was initiated in basal salt medium supplemented with trace salts at pH 5.5. Biomass was accumulated in a batch and a fed batch phase with glycerol as the sole carbon source for about 28 h. Expression of target protein was induced by methanol feeding according to a three-step profile recommended by Invitrogen (“Pichia fermentation process guidelines”). The fermentation process was stopped after 68 h. Afterwards, cells were separated from the medium by centrifugation at 6000×g and 4° C. for 20 min.

Histidine was added to a final concentration of 1 mM and the supernatant was applied in upward flow direction with a flow of 12 ml/min onto an expanded bed absorption column (Streamline column 25 [2.5×22 cm—settled] with Streamline Chelating Sepharose, GE Healthcare, Sweden) saturated with Ni²⁺ ions and equilibrated with 50 mM phosphate buffer, pH 6.8, 300 mM sodium chloride. Bound enzyme was washed with 1.5 L 50 mM phosphate buffer, pH 6.8, 300 mM sodium chloride. Enzyme was eluted in downward flow direction at a flow rate of 8 ml/min using 50 mM phosphate buffer, pH 6.8, 300 mM sodium chloride containing 100 mM histidine. QC-containing fractions were pooled and ammonium sulfate was added to a final concentration of 700 mM. After centrifugation at 100000×g (4° C.) for 1 h the resulting solution was applied onto a Butyl Sepharose fast flow column (1.6×13 cm, GE Healthcare, Sweden) at a flow rate of 2 ml/min. Bound enzyme was washed for 3 column volumes applying 50 mM phosphate buffer, pH 6.8, 700 mM ammonium sulfate and eluted in reversed flow direction with 5 mM phosphate buffer, pH 6.8. Fractions containing QC-activity were pooled and dialysed over night at 4° C. against a 100 fold excess v/v of 30 mM Bis-Tris, pH 6.8. The solution was centrifuged at 100000×g (4° C.) for 1 h and applied onto a Uno Q6 column (12×53 mm, Bio-Rad, Germany) at a flow rate of 4 ml/min. Bound enzyme was washed for 3 column volumes applying 30 mM Bis-Tris, pH 6.8. Enzyme was eluted with 30 mM Bis-Tris, pH 6.8 containing 100 mM sodium chloride. QC-containing fractions were pooled and purity was analysed by SDS-PAGE. The purified enzyme was stored at −20° C. after addition of 50% glycerol or without glycerol at −80° C.

(B) Characterization of Human Glutaminyl Cyclase

Detection of Disulfide Bond Applying SDS-PAGE

For detection of a disulfide bond of hQC WT (wild type) and the C139A/C164A mutant SDS-PAGE was applied. Therefore enzyme was deglycosylated using Endoglycosidase H_(f) (1000 units per 0.2 mg QC for 1 h at room temperature; NEB, Germany) and mixed with loading buffer:

TABLE 2 Buffer conditions for reducing and non-reducing SDS-PAGE Non-reducing conditions Reducing conditions 45 mM Tris-HCl, pH 6.8 45 mM Tris-HCl, pH 6.8   10% glycerol   10% glycerol 0.01% bromine phenol blue 0.01% bromine phenol blue   5% β-mercaptoethanol

Samples were boiled for 5 min and a 15% Tris-Glycine SDS gel (6 cm×8 cm) was run with 5 μg protein per lane at 200 V for 2 h. Protein was visualised by Coomassie staining (FIG. 5).

While migration shifts for the hQC WT between reducing (shaded grey lanes, disulfide bond is broken) and non-reducing (white lanes, disulfide bridge) conditions; due to diffusion of the reducing agent the band pattern of lanes 3 and 6 is a mixed type; no difference in migration appears between both conditions for the C139A/C164A mutant. No reducing events/breaking of a disulfide bond occurs which influences migration on SDS-PAGE for the mutant.

Conformational Stability—CD-Spectra

hQC and the C139A/C164A mutant were desalted by size exclusion chromatography using a Sephadex G-25 fast desalting column (1.0×10 cm; GE Healthcare, Sweden), which was equilibrated in 10 mM phosphate buffer, pH 6.8. Protein concentration was adjusted to 40 μM (WT) and 37 μM (C139A/C164A).

CD-spectra were acquired with a Jasco J-715 spectrapolarimeter (Jasco, Germany) using quartz cuvettes of 0.1 cm pathlength and an external thermostat (Julabo F25, Julabo, Germany). For wavelength scans (general settings: sensitivity 100 mdeg, data pitch 1 nm, scanning mode continuous, scanning speed 50 nm/min, response time 1 s, band with 1 nm) the mean of 10 scans between 190 and 260 nm (far UV/amide region) was calculated and corrected by subtraction of the buffer spectrum (FIG. 6). The percentage of secondary structure elements at 20° C. was calculated using the Jasco secondary structure estimation program based on the method of Yang (Yang, J. T., Wu, C. S., and Martinez, H. M. [1986] Methods Enzymol 130, 208-269).

Both enzymes show a typical CD spectrum for proteins with a high α-helical content characterized by minima at wavelength of 208 nm and 222 nm. The tertiary structure was calculated to WT: 54% α-helix, 25.8% β-sheet, 18.5% random; C139A/C164A: 55.6% α-helix, 25.8% β-sheet, 20.2% random. These values are very similar, leading to the conclusion that the loss of the disulfide bond does not evoke changes in tertiary structure.

To study the conformational stability, temperature was increased from 20 to 80° C. with a velocity of 30° C./h and a delay of 60 s. Wavelength scans were done every 10 K whereas the ellipticity at a wavelength of 227 nm was measured every 2 K (FIG. 7). At this wavelength, a major change of the spectrum could be observed. A sigmoid fit revealed a temperature of 49° C. where half of the WT enzyme is unfolded. For the C139A/C164A variant, this value is decreased to 46° C. A conformation stabilizing effect can be attributed to the disulfide bond of hQC.

Because of the wide-meshed recording, this difference can not be detected in the wavelength scans (FIG. 8). Refolding does not occur upon cooling.

Conformational Stability—Fluorescence Emission Spectra

Fluorescence emission spectra were recorded in the wavelength range between 320 and 400 nm (major emission caused by tryptophan residues) at 22° C. using the luminescence spectrometer LS 50 B (Perkin-Elmer, USA). The excitation wavelength was 295 nm (only excitation of tryptophan residues) at a scanning speed of 100 nm/min using a slit width of 3 nm for excitation and 6 nm for emission. To study conformational stability hQC WT and the C139A/C164A mutant were diluted with 50 mM phosphate buffer, pH 6.8 containing diverse concentrations of guanidine hydrochloride (GdmCl) to a concentration of 0.24 μM (WT) respectively 0.31 μM (C139A/C164A mutant). Spectra were acquired and the spectrum of the corresponding buffer was subtracted (FIG. 9).

Usually, upon denaturation the emission maximum of proteins exhibits a tryptophan-mediated shift from a shorter wavelength to about 350 nm which corresponds to the fluorescence maximum of tryptophan in aqueous solutions (Schmid, F. X. [1989] Protein Structure: A Practical Approach [Creighton, T. E:, Ed.] pp 251-285, IRL Press, oxford). The native enzymes (hQC WT and C139A/C164A mutant) exhibit a fluorescence emission maximum at 344 nm which shifts upon unfolding to a wavelength of 354 nm, indicating a more hydrophilic environment of the tryptophan residues compared to the folded state.

The conformation of both enzymes is stable up to a GdmCl concentration of 2M. At a concentration of 2.5 M unfolding can be detected indicated by a reduction of fluorescence emission. In comparison to hQC WT the unfolding is increased for the C139A/C164A mutant. While there is still a folded fraction at 4 M GdmCl for hQC WT the C139A/C164A mutant is completely unfolded.

At an emission wavelength of 337 nm major changes in relative fluorescence could be observed. The GdmCl concentrations where half of the protein is unfolded could be determined as 3.2 M for hQC WT and 2.9 M for the C139A/C164A mutant (FIG. 10). This confirms the conformation stabilizing effect of the disulfide bond.

(C) Crystallization of Human Glutaminyl Cyclase

Crystallisation was conducted by applying the hanging drop vapour diffusion method. First screening for crystallisation conditions was carried out with several commercial available basic screening kits at 13° C. (protein concentration 5 mg/ml, 0.5 mM Inhibitor A) and 21° C. (protein concentration 7.5 mg/ml, 0.5 mM Inhibitor A). The most promising crystals grew in buffer C5 of the JBScreen Classic 7 kit by Jena Bioscience (JBS, Germany) [30% (w/v) 2-methyl-2,4-pentanediol; 10% (w/v) PEG 4000; 100 mM imidazole-HCl, pH 8] at a temperature of 21° C. and a protein concentration of 7.5 mg/ml. For further optimization the concentration of PEG 4000 was varied revealing a concentration of 11% (w/v) PEG 4000 as ideal. By X-ray structure analysis a resolution of 2.1 Å was achieved.

Crystal Growth

Crystals were grown using the hanging drop vapor diffusion technique at room temperature (21° C.) in Easyxtal 24-well plates (Qiagen). The mother liquor buffer consisted of 0.1M imidazol pH 8, 30% (v/v) 2-methyl 2,4-pentanediol and 11% (w/v) 4000PEG. Protein solution concentrated up to a 10 mg/ml was recombinant human glutaminyl cyclase in the presence of 25 mM Bis-Tris pH6.8/100 mM NaCl buffer. A mixture of 1 μl mother liquor buffer and 1 μl protein solution was set. Crystals appeared usually ten to fifteen days after the experiment was initiated and they displayed a macroscopic plate shape (FIG. 11).

Cryosolution

Before x-ray measurements, crystals were rapidly soaked into a new cryo-buffer solution where mother liquor buffer solution was saturated with 15% (v/v) glycerol. Immediately crystals were collected from the cryo-buffer solution using a pin nylon loop and they were mounted onto the goniometer and flash frozen at −180° C. under a nitrogen stream.

Data Collection

Data collection from a single crystal was undertaken by means of Cu Kα radiation (λ=1.5418 Å) by using a rotating-anode source (RA Micro 007; Rigaku/MSC, Tokyo, Japan) and CCD detector device (CCD Saturn 944+, Rigaku) with Varimax™ Optics (Rigaku) and an AFC-11 goniometer.

Finally, the crystal was demounted and stored in liquid nitrogen for an additional measurement in the beam line BL14.1 of the synchrotron in Berlin (Bessy).

Data Processing

Data processing was carried out using the programs included in the crystallographic suite CCP4. The image reflection intensities were indexed and integrated using the program Mosflm. Afterwards the integrated intensities were scaled and merged using the program Scala. Moreover, initial phases were obtained with a molecular replacement approach using Phaser and taking the crystal structure with PDBid 2AFO as searching model. Thus, initial electron densities were obtained. Subsequent manual building of the missing fragments and maximum-likelihood refinement cycles were performed by using the programs COOT and REFMAC5 as well as from the CCP4 suite.

Results

Table 3 summarizes the statistics of the data set obtained in Berlin synchrotron Bessy with a crystal of the human glutaminyl cyclase and their corresponding statistics for the data processing and model building. Programs used were for data processing MOSFLM and SCALA, for molecular replacement PHASER, for refinement REFMAC5 and for Model Building: Coot (all of them included in the CCP4 suite). Numbers between brackets belong to the outer shell resolution limit.

TABLE 3 Summary of Statistics for Human Glutaminyl Cyclase Crystal Data collection Data set collected at BESSY Space group C121 Cell dimensions? a, b, c (Å) 82.6 63.9 77.5 α, β, γ (°) 90.0 105.7 90.0 Resolution (Å) 11.98-2.08 Rmerge 8.0 (51.3) I/SigmaI 8.6 (2.0)  Completeness (%) 94.4 Redundancy 2.9 Refinement Resolution (Å) 11.98-2.08 No. reflections (work/test) 22217/1073  R_(work)/R_(free) 20.5/26.3 No. atoms Protein 2556 Water 167 B-factors (Mean value) 29.64 R.m.s deviations Bond lengths (Å) 0.022 Bond angles (°) 1.915

Overall three dimensional structure. The model built from the obtained experimental electron density includes the human glutaminyl cyclase residues from A³⁵ to L³⁶¹, a zinc ion and an imidazole molecule. The protein shows a globular α/β hydrolase fold (FIG. 12). A central β-sheet is formed with six β-strands all in parallel fashion but not the second. This β-sheet is surrounded by α-helices in a sandwich manner with two helices in one side and six more α-helices in the opposite face. The protein's structure is completed with a rather large amount of random coiled loops which are building the active site of the enzyme. This active site is accommodating a zinc ion which is coordinated by three protein residues, E²⁰², D¹⁵⁹ and H³³⁰ and an imidazol molecule (FIG. 13). Moreover the protein shows the presence of a disulfide bridge between residues C¹³⁹ and C¹⁶⁴. Additionally four cis-peptide bonds are present between the pair of residues: D¹⁵⁹-S¹⁶⁰, H²²⁸—P²²⁹, G³⁰¹-V³⁰² and S³²³—P324. Finally two segments of the polypeptide chain are not visible in the electron density. The gaps include residues between K¹⁸² and D¹⁹⁰ and between F¹⁴⁶ and N¹⁵⁰.

Example 2 Isolation, Purification and Crystallisation of Murine Glutaminyl Cyclase

(A) Isolation and Purification of Murine Glutaminyl Cyclase

Cloning Procedures

The primers for isolation of the open reading frame of mQC were designed using PubMed nucleotide entry AK017598, encoding the putative mQC. The primer sequences were as follows: sense 5′-ATATGCATGCATGGCAGGCAGCGAAGACAAGC-3′ (SEQ ID NO: 9); antisense 5′-ATATAAGCTTTTACAAGTGAAGATATTCCAACACAAAGAC-3′ (SEQ ID NO: 10). Total RNA was isolated from murine insulinoma cell line β-TC 3 cells using the RNeasy Mini Kit (Qiagen, Germany) and reversely transcribed by SuperScriptII (Invitrogen, Germany). Subsequently, mQC cDNA was amplified on a 1:12.5 dilution of generated product in a 50 μl reaction with Herculase Enhanced DNA-Polymerase (Stratagene, USA), inserted into the PCR Script CAM Cloning vector (Stratagene, USA) and verified by sequencing. The cDNA fragment encoding the mature mQC was amplified using the primers 5′-ATACTCGAGAAAAGAGCCTGGACGCAGGAGAAG-3′ (XhoI, sense) (SEQ ID NO: 11) and 5′-ATATCTAGATTACAAGTGAAGATATTCCAAC-3′ (XbaI, antisense) (SEQ ID NO: 12). The digested fragment was ligated into the vector ρPICZαB, propagated in E. coli and verified by sequencing of the sense and antisense strand. Finally, the expression plasmid was linearized using PmeI, precipitated, and stored at −20° C.

Transformation of P. pastoris and Mini-Scale Expression

1-2 μg of plasmid DNA were used for transformation of competent P. pastoris cells by electroporation according to the manufacturer's instructions (BioRad, Germany). Selection was conducted on plates containing 100 μg/ml Zeocin. In order to test the recombinant yeast clones upon mQC expression, recombinants were grown for 24 h in 10 ml conical tubes containing 2 ml BMGY. Subsequently, the yeast was centrifuged and resuspended in 2 ml BMMY containing 0.5% methanol. This concentration was maintained by addition of methanol every 24 h for about 72 h. Subsequently, QC activity in the supernatant was determined. Clones that displayed the highest activity were chosen for further experiments and fermentation.

Large-Scale Expression and Purification of murine QC (mQC)

The large-scale expression of mQC was performed in a 5 L reactor (Biostad B; B. Braun Biotech, Germany). Fermentation was carried out in basal salt medium supplemented with trace salts at pH 5.5. Initially, biomass was accumulated in batch phase with glycerol as sole carbon source for about 28 h. The expression of mQC was initiated by methanol feeding according to a three-step profile recommended by Invitrogen for an entire fermentation time of approximately 65 h.

Subsequently, cells and turbidity were removed by two sequential centrifugation steps at 6000×g and 38000×g for 20 min and 3.5 h. For purification, the fermentation broth was diluted with cold water to a conductivity of about 4 mS/cm and applied in reversed flow direction (12 ml/min) onto a Streamline SP XL column (2.5×100 cm), equilibrated with 0.05 M phosphate buffer, pH 6.4. After a washing step in reversed flow direction with equilibration buffer for 2 column volumes, proteins were eluted at a flow rate of 8 ml/min using 0.15 M Tris-HCl buffer, pH 7.6, containing 1.5 M NaCl in forward direction. The QC containing fractions were pooled, and ammonium sulfate was added to a final concentration of 1 M. The resulting solution was centrifuged at 75000×g for 20 min and applied onto a butyl-Sepharose FF column (1.6×13 cm) at a flow rate of 4 ml/min, equilibrated with 0.05 M phosphate buffer pH 6.8, containing 1 M ammonium sulfate. Bound mQC was washed with 0.05 M phosphate buffer, pH 6.8, containing 1 M ammonium sulfate for 5 column volumes and eluted in reversed flow direction with 0.05 M phosphate buffer, pH 6.8. The fractions containing mQC were pooled and diluted with water to a conductivity lower than 10 mS/cm and applied onto an Uno S column (BioRad, Germany), equilibrated with 0.05 M phosphate buffer, pH 6. After a washing step using equilibration buffer containing 0.09 M NaCl, mQC was eluted using the same buffer containing 1.5 M NaCl. The fractions exhibiting QC activity were pooled and applied to a HiPrep desalting column (2.6×10 cm), which was equilibrated with 0.015 M BisTris, pH 6.8, containing 0.1 M NaCl. After purification the mQC was concentrated to 10 mg/ml and stored at −80° C.

(B) Crystallization of Murine Glutaminyl Cyclase

Crystal Growth

Crystals were grown using the hanging drop vapor diffusion technique at room temperature (21° C.) in Easyxtal 24-well plates (Qiagen). The mother liquor buffer consisted of 0.1M sodium acetate pH 5.3, 0.2M ammonium sulphate and 12% (w/v) 2000 MME-PEG. The protein solution of recombinant mouse glutaminyl cyclase (mQC) was concentrated up to 10 mg/ml in the presence of 15 mM Bis-Tris pH6.8/100 mM NaCl buffer. A mixture of 1 μl mother liquor buffer and 1 μl protein solution was set. Crystals appeared usually in the course of one week after experiment was initiated and they displayed a macroscopic rod/needle shape (FIG. 14).

Cryosolution

Before x-ray measurements, crystals were rapidly soaked into a new cryo-buffer solution where mother liquor buffer solution was saturated with 20% (v/v) glycerol. Immediately crystals were collected from the cryo-buffer solution using a pin nylon loop and they were mounted onto the goniometer and flash frozen at −180° C. under a nitrogen stream.

Data Collection

Data collection from a single crystal was undertaken by means of Cu Kα radiation (λ=1.5418 Å) by using a rotating-anode source (RA Micro 007; Rigaku/MSC, Tokyo, Japan) and CCD detector device (CCD Saturn 944+, Rigaku) with Varimax™ Optics (Rigaku) and an AFC-11 goniometer.

Finally the crystal was demounted and stored in liquid nitrogen for an additional measurement in the beam line Bessy-MX BL14.1 of the synchrotron in Berlin (Bessy) with a wave length radiation (λ=0.91841 Å)

Data Processing

Data processing was carried out as described for Example 1.

Results

Overall three dimensional structure. Three different protein-inhibitor complexes were obtained. The models built from the obtained experimental electron density include the mouse glutaminyl cyclase residues from A³⁶ to L³⁶², a zinc ion and an inhibitory molecule. The protein shows a globular α/β hydrolase fold (FIG. 15). A central β-sheet is formed with six β-strands all in parallel fashion but not the second. This β-sheet is surrounded by α-helices in a sandwich manner with two helices in one side and six more α-helices in the opposite face. The protein's structure is completed with a rather large amount of random coiled loops which are building the active site of the enzyme (FIGS. 16, 18 and 19). This active site is accommodating a zinc ion which is coordinated by three protein residues D¹⁶⁰, E²⁰³ and H³³¹ and an inhibitor molecule. Three different compounds were crystallized in different crystallization set ups, Inhibitor A, Inhibitor C and Inhibitor B, respectively FIGS. 16, 18 and 19. These three different molecules displayed the same localization and overall interaction patterns. Moreover the protein shows the presence of a disulfide bridge between residues C¹⁴⁰ and C¹⁶⁵ (FIG. 17).

Table 4 summarizes the statistics of the data set obtained with the crystals of mouse glutaminyl cyclase and their corresponding data processing and model building. The programs used were for data processing MOSFLM and SCALA, for molecular replacement PHASER for refinement REFMAC5 and for Model Building Coot. All programs belong to the CCP4 suite. Numbers between brackets belong to the outer shell resolution limit. On the table header are indicated the co-crystallized inhibitor for their respective summary of statistics of each data set.

TABLE 4 Summary of Statistics for Murine Glutaminyl Cyclase Crystals Data collection Inhibitor A Inhibitor B Inhibitor C Data ser collected at BESSY BESSY BESSY Space group P212121 P212121 P212121 Cell dimensions? a, b, c (Å) 42.7 83.0 95.7 42.7 84.6 96.5 42.7 84.6 97.2 α, β, γ (°) 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 Resolution (Å) 19.81-1.80 32.0-1.98 30.7-1.9 Rmerge 14.3 (73.1) 7.3 (59.0) I/SigmaI 14.3 (2.4)  17.5 (2.4)  Completeness (%) 100.0 100.0 100.0 Redundancy 4.0 (3.4) 3.1 (1.6)  Refinement Resolution (Å) 19.81-1.80 32.0-1.98 30.7-1.9 No. reflections 29995/1580  23586/1242  20392/1074  (work/test) R_(work)/R_(free) 18.98/25.44 18.0/23.8 17.6/22.8 No. atoms Protein 2622 2622 2622 Water 340 318 285 B-factors (Mean value) 21.00 24.99 24.99 R.m.s deviations Bond lengths (Å) 0.015 0.018 0.015 Bond angles (°) 1.637 2.175 1.747 

What is claimed is:
 1. A co-crystal comprising murine glutaminyl cyclase and a glutaminyl cyclase inhibitor selected from the group consisting of inhibitor A, inhibitor B, and inhibitor C, the co-crystal having a characterised space group of P212121 and having unit cell dimensions of +/−5% of a=42.7 Å, b=84.0 Å, c=96.5 Å, α=90.0°, β=90.0° and γ=90.0°; wherein, the murine glutaminyl cyclase comprises amino acid residues A36 to L362 of SEQ ID NO: 13; inhibitor A has a structure of

inhibitor B has a structure of

inhibitor C has a structure of


2. A co-crystal as defined in claim 1, having unit cell dimensions of a=42.7 Å, b=83.0 Å, c=95.7 Å, α=90.0°, β=90.0° and γ=90.0°, wherein the glutaminyl cyclase inhibitor is inhibitor A.
 3. A co-crystal as defined in claim 1, having unit cell dimensions of a=42.7 Å, b=84.6 Å, c=96.5 Å, α=90.0°, β=90.0° and γ=90.0°, wherein the glutaminyl cyclase inhibitor is inhibitor B.
 4. A co-crystal as defined in claim 1, having unit cell dimensions of a=42.7 Å, b=84.6 Å, c=97.2 Å, α=90.0°, β=90.0° and γ=90.0°, wherein the glutaminyl cyclase inhibitor is inhibitor C.
 5. A co-crystal as defined in claim 1, which diffracts x-rays for determination of atomic co-ordinates of the crystal to a resolution of between 1.9 Å and 32.0 Å.
 6. A co-crystal as defined in claim 1, wherein the murine glutaminyl cyclase consists of amino acid residues A36 to L362 of SEQ ID NO:13.
 7. A co-crystal as defined claim 1, which comprises a binding pocket provided by residues D160, E203 and H331 of SEQ ID NO: 13 according to the coordinates of FIGS. 2 to
 4. 8. A co-crystal as defined in claim 6, which comprises a disulfide bridge between residues C140 and C165 of SEQ ID NO:
 13. 9. A co-crystal as defined in claim 7, which comprises a disulfide bridge between residues C140 and C165 of SEQ ID NO:
 13. 10. A co-crystal as defined in claim 1, which comprises a zinc ion coordination site provided by residues D160, E203, and H331.
 11. A co-crystal as defined in claim 1, wherein the co-crystal comprises a zinc ion coordinated by residues D160, E203, and H331.
 12. A method of preparing the co-crystal of murine glutaminyl cyclase and a glutaminyl cyclase inhibtor as described in claim 1, which comprises the steps of: (a) providing a solution of murine glutaminyl cyclase, in the presence of a glutaminyl cyclase inhibitor selected from the group consisting of inhibitor A, inhibitor B, and inhibitor C, in a suitable buffer such as 15 mM Bis-Tris pH6.8/100 mM NaCl buffer; (b) mixing the solution with a crystallization solution comprising 0.1M sodium acetate pH 5.3, 0.2M ammonium sulphate and 12% (w/v) 2000 MME-PEG; and (c) incubating the mixture under conditions to promote hanging drop vapor diffusion for a time sufficient to produce the co-crystal of murine glutaminyl cyclase and the glutaminyl cyclase inhibitor.
 13. A co-crystal comprising murine glutaminyl cyclase and a glutaminyl cyclase inhibitor obtainable by the crystallisation method as defined in claim
 12. 